1. Field of the Invention
The present invention relates to methods for producing fertile transgenic Impatiens plants, to fertile transgenic Impatiens plants, and to transgenic seeds and progeny thereof. In particular, this invention is directed to transgenic Impatiens that express at least one macromolecule that confers resistance to Impatiens pathogens, confer improved tolerance to environmental stresses, or otherwise enhance the commercial value of the plant.
2. Background
The Impatiens genus is a member of the family is Balsaminaceae and comprises some five hundred to six hundred species, many of which are commercially cultivated as ornamental plants. Impatiens include plants originating from Africa, New Guinea, Celebes and Java. Grey-Wilson, IMPATIENS OF AFRICA (A. A. Balkema 1980); H. F. Winters, Am. Hotic., 52, 923 (1973). The Impatiens from Africa, India and the South Pacific include respectively, I. wallerana, I. balsamina, I. hawkeri. Among these I. wallerana, also known as I. sultani or I. holstii, is probably the most commonly grown. I. wallerana comprises the largest market share of all bedding plants sold in the United States and therefore is an important horticultural crop.
The New Guinea Impatiens (NGI) encompasses a group of interbreeding species that include I. schlecteri Warb., I. herzogii K.Schum, I. linearifolia Warb., I. mooreana Schltr., I. hawkeri Bull, and other species of the same geographic origin which are interfertile. Java and Celebes Impatiens are known as I. platypetala Lindl. and I. platylpetala aurantiaca Steen, respectively. K. Han et al., Scientia Horticulturae, 32, 307 (1987).
Insect pests and diseases caused by pathogens can kill Impatiens even under greenhouse conditions. Illustrative insect pests include whiteflies, mealybugs, thrips, aphids, and spider mites. Impatiens are also susceptible to diseases caused by fungi. Fungal infestation include infections by Rhizoctonia and Pythium, which can cause stunting or death of Impatiens. Impatiens is also susceptible to Botrytis blight, and infection by Alternaria and Fusarium.
Furthermore, Impatiens is also subject to bacterial infection such as Pseudomonas infection. As for viral pathogens, Impatiens is primarily susceptible to infection by the tospovirus, Impatiens necrotic spot virus (INSV), but also is a known host for the related tospovirus, tomato spotted wilt virus (TSWV). Impatiens are also known to be hosts to tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and tobacco streak virus (TSV).
Although chemical treatment can control certain of these insect pests and disease pathogens, such treatment can also have an adverse effect upon Impatiens. An alternative to chemical treatment is to genetically engineer transgenic Impatiens that express polypeptides capable of protecting the plant against the insects and pathogens. The production of transgenic plants can further be used to enhance the commercial value of Impatiens by conferring resistance to environmental stresses, such as, drought, salinity, heat, cold, frost, and sun. The production of transgenic plants can further be used to enhance the commercial value of Impatiens by controlling characteristics such as flower color, leaf color, flower size and pattern, early flowering, day neutrality, free branching, dwarfness, fragrance, among others. Other desired qualities include bioluminescence, seedling and plant vigor, and flower doubleness.
Accordingly, there is a need for a method to introduce foreign genes into Impatiens to confer resistance to INSV, impart fragrance or drought tolerance, as well as other desired properties. However, there has been no report to date of the successful production of transgenic Impatiens.
On the contrary, although researchers report the isolation of genes conferring resistance to tospoviruses in general, and INSV, in particular, these references do not disclose transformation of Impatiens. For instance WO 96/29420 to De Haan, describes transgenic plants resistance to Tospovirus, but discloses only transgenic tobacco. Similarly, EP 0566525 to Van Grinsven et al., discloses DNA constructs to transform plants to achieve resistance to INSV, but discloses only Nicotiana tabacum and Petunia hybrida transformation.
Similarly, WO 95/24486 to Attenborough, et al., discloses antimicrobial proteins isolated from seed of Impatiens and transgenic plants produced from DNA constructs which encode such proteins. This reference, however, fails to report successful transformation of Impatiens disclosing instead only tobacco transformation.
Furthermore, other researchers have described Impatiens as a desirable plant for transformation with, for example, pigment-inducing DNA constructs, EP 0524910 to Van Holst et al., or phytochrome polypeptide-encoding constructs, EP 0354687 to Hershey et al. These references do not disclose, however, actual transformation of Impatiens.
Finally, Takeshi et al., Shokubutsu Soshiki Baiyo 12: 73 (1995); Chem. Abs. 122(25) 310945u, report transient gene expression in I. balsamina and I. wallerana mature pollen transformed with plasmid pBI221 via a N-2 laser method. This reference, however, fails to report stable integration of the DNA plasmid construct, or production of a transformed Impatiens plants.
Thus, a need exists for a method to stably introduce foreign genes into Impatiens to enhance viral resistance, drought resistance, and impart fragrance as well as other traits that enhance the commercial value of this important ornamental crop.
Accordingly, it is an object of the present invention to provide a method to produce transgenic Impatiens.
It is a further object of this invention to provide transgenic Impatiens that express at least one macromolecule that confers protection against disease causing pathogens. These and other objects are achieved in accordance with one embodiment of the present invention by the provision of a method for producing transgenic Impatiens plants, comprising the steps of:
(a) introducing an expression vector into a tissue explant medium to produce transformed explant, wherein said expression vector comprises a selectable marker gene and a second foreign gene, or
(axe2x80x2) introducing two expression vectors into said tissue explant to produce transformed explant, wherein one of said expression vectors comprises a selectable marker gene, and wherein the second of said expression vectors comprises a second foreign gene;
(b) culturing said transformed explant on a selection medium;
(c) culturing said transformed explant on regeneration medium; and
(d) recovering fertile transgenic plants from the transgenic explants capable of transmitting the foreign gene to progeny.
Also provided is a method of Impatiens transformation wherein the tissue explant is pre-cultured prior to introducing the expression vector in the explant wherein the explant is pre-cultured in MS medium comprising from approximately 0.5 mg/L to 2 mg/L TDZ followed by MS medium containing auxin and cytokinin, preferably approximately 0.05 to 0.2 mg/L NAA and approximately 1 to 6 mg/L Zeatin.
Also provided is a fertile transgenic Impatiens plant having stably integrated in the plant genome a foreign. gene, wherein the transgenic plant is capable of transmitting the foreign gene to progeny.